角膜内皮细胞玻璃化冷冻保护剂的实验研究  

Cryoprotectants for the Vitrification of Corneal Endothelial Cells

作　　者：范文霞[1] 马学虎[1] 葛丹[1] 于小川[1] 刘天庆[1] 崔占峰[2] 

机构地区：[1]大连理工大学化工学院,大连市干细胞与组织工程研发中心,辽宁大连116024 [2]牛津大学工程科学系,英国牛津

出　　处：《高校化学工程学报》2009年第5期845-851,共7页Journal of Chemical Engineering of Chinese Universities

基　　金：国家自然科学基金合作项目(2002008);大连市科技计划项目(2005E11SF068)

摘　　要：对角膜内皮细胞的玻璃化冷冻保护剂进行了研究,进而获得一种玻璃化溶液。首先筛选渗透性保护剂;在此基础上,进行糖类非渗透性保护剂的筛选。渗透性保护剂的筛选范围包括二甲基亚砜、乙二醇、1,2-丙二醇、2,3-丁二醇、乙酰胺和乙二醇单甲醚。糖类非渗透性保护剂包括木糖、果糖、甘露糖、葡萄糖、麦芽糖、蔗糖和海藻糖。所采用的细胞活性的评价方法包括台盼兰拒染法和CCK-8试剂盒。研究结果表明:在众多的渗透性保护剂中,乙二醇的保护效果最好;糖类保护剂中,葡萄糖的保护效果较好。获得的玻璃化溶液的组成是:载体溶液-乙二醇-葡萄糖,其中乙二醇和葡萄糖的浓度分别为52%(w/w)和8%(w/w)。经此玻璃化溶液冷冻保存后的角膜内皮细胞的活率高达(85.5±0.7)%。This work was to select and test the possible cryoprotective agents （CPAs） and to obtain a suitable formula for vitrification of corneal endothelial cells （CECs）. Fresh bovine CECs were isolated and tested according to an optimized vitrification protocol with multi-step CPA loading and removal. Two types of CPA components,i.e. the penetrating CPA components and nonpenetrating saccharide CPAs were experimentally evaluated via using the viability measured by trypan blue. Dimethyl sulfoxide （Me2SO）,ethylene glycol （EG）,1,2-propanediol （1,2-PD）,2,3-butanediol （2,3-BD）,acetamide （Ace） and ethylene glycol monomethyl ether （EGMME） were chosen as the penetrating CPA components,and xylose,fructose,mannose,glucose,maltose,sucrose and trehalose were used as the saccharide CPAs,respectively. The results show that EG is the most suitable penetrating CPA component and glucose the best saccharide CPA. The optimized concentrations for each component in the chosen vitrification solution are 52% EG and 8% glucose. The CEC survival ratio of （85.5±0.7）% （mean±S.D.） was obtained following the established protocol.

关 键 词：角膜内皮细胞 玻璃化 保护剂 玻璃化溶液 

分 类 号：R318.52[医药卫生—生物医学工程]